實(shí)用標(biāo)準(zhǔn)文案沉淀蛋白質(zhì)的常用方法（ TCA、乙醇、丙酮沉淀蛋白操作步驟）2010-08-1815:19TCA-DOCForprecipitationofverylowproteinconcentrationToonevolumeofproteinsolution,add1/100vol.of2%DOC(Nadeoxycholate,detergent).Vortexandletsitfor30minat4oC.Add1/10ofTrichloroaceticacid(TCA)100%vortexandletsitONat4oC(preparationof100%TCA:454mlH2O/kgTCA.Maintainindarkbottleat4oC.Becareful,usegloves!!!).Spin15min4oCinmicrofugeatmaximumspeed(15000g).Carefullydischargesupernatantandretainthepellet:drytubebyinversionontissuepaper(pelletmaybedifficulttosee).[OPTION:Washpellettwice repelletsamples5minatfullspeedbetweenwashes].Drysamplesundervaccum(speedvac)ordryair.ForSDS,resuspendsamplesinaminimalvolumeofsamplebuffer.(ThepresenceofsomeTCAcangiveayellowcolourasaconsequenceoftheacidificationofthesamplebuffer;titratewith1NNaOHor1MTrisHClpH8.5toobtainthenormalbluesamplebuffercolour.)精彩文檔實(shí)用標(biāo)準(zhǔn)文案NormalTCAToeliminateTCAsolubleinterferencesandproteinconcentration1)ToasampleofproteinsolutionaddTrichloroaceticacid(TCA)100%toget13%finalconcentration.Mixandkeep5min –20oCandthen15min4oC;orlongertimeat4oCwithoutthe –20oCstepforlowerproteinconcentration.Suggestion:leaveONiftheproteinconcentrationisverylow.(preparationof100%TCA:454mlH2O/kgTCA.Maintainindarkbottleat4oC.Becareful,usegloves!!!).Spin15min4oCinmicrofugeatmaximumspeed(15000g).Carefullydischargesupernatantandretainthepellet:drytubebyinversionontissuepaper(pelletmaybedifficulttosee).ForSDS,resuspendsamplesinaminimalvolumeofsamplebuffer.(ThepresenceofsomeTCAcangiveayellowcolourasaconsequenceoftheacidificationofthesamplebuffer;titratewith1NNaOHor1MTrisHClpH8.5toobtainthenormalbluesamplebuffercolour.)AcetonePrecipitationToeliminateacetonesolubleinterferencesandproteinconcentration1)Add1volumeofproteinsolutionto4volumesofcoldacetone.Mixandkeepatleast20min –20oC.(Suggestion:leaveONifthe精彩文檔實(shí)用標(biāo)準(zhǔn)文案proteinconcentrationisverylow).Spin15min4oCinmicrofugeatmaximumspeed(15000g).Carefullydischargesupernatantandretainthepellet:drytubebyinversionontissuepaper(pelletmaybedifficulttosee).Drysamplesundervaccum(speed-vac)ordryairtoeliminateanyacetoneresidue(smelltubes).ForSDS,resuspendsamplesinaminimalvolumeofsamplebuffer.EthanolPrecipitationUsefulmethodtoconcentrateproteinsandremovalofGuanidineHydrochloridebeforeSDS1)Addto1volumeofproteinsolution9volumesofcoldEthanol100%.Mixandkeepatleast10min.at –20oC.(Suggestion:leaveON).2)Spin15min4oCinmicrocentrifugeatmaximumspeed(15000g).Carefullydischargesupernatantandretainthepellet:drytubebyinversionontissuepaper(pelletmaybedifficulttosee).3)Washpelletwith90%coldethanol(keepat–20oC).Vortexandrepelletsamples5minatfullspeed.4)Drysamplesundervaccum(speedvac)ordryairtoeliminateanyethanolresidue(smelltubes).ForSDS,resuspendsamplesinaminimalvolumeofsamplebuffer.精彩文檔實(shí)用標(biāo)準(zhǔn)文案TCA-DOC/AcetoneUsefulmethodtoconcentrateproteinsandremoveacetoneandTCAsolubleinterferences1.Toonevolumeofproteinsolutionadd2%Nadeoxycholate(DOC)to0.02%final(for100 μlsample,add1 μl2%DOC).Mixandkeepatroomtemperatureforatleast15min.100%trichloroaceticacid(TCA)toget10%finalconcentration(preparationof100%TCA:454mlH2O/kgTCA.Maintainindarkbottleat4oC.Becareful,usegloves!!!).Mixandkeepatroomtemperatureforatleast1hour.Spinat4oCfor10min,removesupernatantandretainthepellet.Drytubebyinversionontissuepaper.Add200μloficecoldacetonetoTCApellet.Mixandkeeponiceforatleast15min.Spinat4oCfor10mininmicrocentrifugeatmaximumspeed.Removesupernatantasbefore(5),dryairpellettoeliminateanyacetoneresidue(smelltubes).ForSDS,resuspendsamplesinaminimalvolumeofsamplebuffer.(ThepresenceofsomeTCAcangiveayellowcolourasaconsequenceoftheacidificationofthesamplebuffer;titratewith精彩文檔實(shí)用標(biāo)準(zhǔn)文案1NNaOHor1MTrisHClpH8.5toobtainthenormalbluesamplebuffercolour.)AcidifiedAcetone/MethanolUsefulmethodtoremoveacetoneandmethanolsolubleinterferenceslikeSDSbeforeIEF1)Prepareacidifiedacetone:120mlacetone+10 Cl(1mMfinalμHconcentration).Prepareprecipitationreagent:Mixequalvolumesofacidifiedacetoneandmethanolandkeepat-20oC.Toonevolumeofproteinsolutionadd4volumesofcoldprecipitationreagent.MixandkeepONat-20oC.Spin15min4oCinmicrofugeatmaximumspeed(15000g).Carefullydischargesupernatantandretainthepellet:drytubebyinversionontissuepaper(pelletmaybedifficulttosee).Drysamplesundervaccum(speed-vac)ordryairtoeliminateanyacetoneormethanolresidue(smelltubes).TCA-EthanolPrecipitationUsefulmethodtoconcentrateproteinsandremovalofGuanidineHydrochloridebeforeSDS精彩文檔實(shí)用標(biāo)準(zhǔn)文案1)Dilute10- 25μlsamplesto100 μlwithH2OAdd100μlof20%trichloroaceticacid(TCA)andmix(preparationof100%TCA:454mlH2O/kgTCA.Maintainindarkbottleat4oC.Becareful,usegloves!!!).Leaveinicefor20min.Spinat4oCfor15mininmicrocentrifugeatmaximumspeed.Carefullydischargesupernatantandretainthepellet:drytubebyinversionontissue(pelletmaybedifficulttosee).4)Washpelletwith100 -μcoldiceethanol,dryandresuspendinsamplebuffer.5)IncasetherearetracesofGuHClpresent,samplesshouldbeloadedimmediatelyafterboilingfor7minat95 °C(ThepresenceofsomeTCAcangiveayellowcolourasaconsequenceoftheacidificationofthesamplebuffer;titratewith1NNaOHor1MTrisHClpH8.5toobtainthenormalbluesamplebuffercolour.)PAGEprepTMProteinClean-upandEnrichmentKit-PIERCEThePAGEprep?Kitenablesremovalofmanychemicalsthatinterfere精彩文檔實(shí)用標(biāo)準(zhǔn)文案withSDSanalysis:guanidine,ammoniumsulfate,othercommonsalts,acidsandbases,detergents,dyes,DNA,RNA,andlipids.PIERCE:#26800-PAGEprepTMProteinClean-upandEnrichmentKit(pdf)ChloroformMethanolPrecipitationUsefulmethodforRemovalofsaltanddetergentsTosampleofstartingvolume100ulAdd400ulmethanolVortexwellAdd100ulchloroformVortexAdd300ulH2OVortexSpin1minute@14,0000gRemovetopaqueouslayer(proteinisbetweenlayers)Add400ulmethanolVortexSpin2minutes@14,000g精彩文檔實(shí)用標(biāo)準(zhǔn)文案RemoveasmuchMeOHaspossiblewithoutdisturbingpelletSpeed-VactodrynessBringupin2XsamplebufferforPAGEReference:Wessel,D.andFlugge,U.I.Anal.Biochem.(1984)138,141-143哈哈,我做過這個(gè)論文哈!配膠緩沖液系統(tǒng)對電泳的影響？在SDS－PAGE不連續(xù)電泳中，制膠緩沖液使用的是 Tris－HCL緩沖系統(tǒng)，濃縮膠是pH6.7，分離膠pH8.9；而電泳緩沖液使用的 Tris－甘氨酸緩沖系統(tǒng)。在濃縮膠中，其pH環(huán)境呈弱酸性，因此甘氨酸解離很少，其在電場的作用下，泳動效率低；而 CL離子卻很高，兩者之間形成導(dǎo)電性較低的區(qū)帶，蛋白分子就介于二者之間泳動。由于導(dǎo)電性與電場強(qiáng)度成反比， 這一區(qū)帶便形成了較高的電壓剃度，壓著蛋白質(zhì)分子聚集到一起， 濃縮為一狹窄的區(qū)帶。當(dāng)樣品進(jìn)入分離膠后，由于膠中 pH的增加，呈堿性，甘氨酸大量解離，泳動速率增加，直接緊隨氯離子之后，同時(shí)由于分離膠孔徑的縮小， 在電場的作用下，蛋白分子根據(jù)其固有的帶電性和分子大小進(jìn)行分離。所以，pH對整個(gè)反應(yīng)體系的影響是至關(guān)重要的，實(shí)驗(yàn)中在排除其他因素之后仍不能很好解決問題的情況，應(yīng)首要考慮該因素。2.樣品如何處理？根據(jù)樣品分離目的不同，主要有三種處理方法：還原 SDS處理、非還原SDS處理、帶有烷基化作用的還原 SDS處理。精彩文檔實(shí)用標(biāo)準(zhǔn)文案1）還原SDS處理：在上樣buffer 中加入SDS和DTT（或Beta巰基乙醇）后，蛋白質(zhì)構(gòu)象被解離，電荷被中和，形成 SDS與蛋白相結(jié)合的分子，在電泳中，只根據(jù)分子量來分離。一般電泳均按這種方式處理，樣品稀釋適當(dāng)濃度，加入上樣Buffer，離心，沸水煮 5min，再離心加樣。2）帶有烷基化作用的還原 SDS處理：碘乙酸胺的烷基化作用可以很好的并經(jīng)久牢固的保護(hù) SH基團(tuán)，得到較窄的譜帶；另碘乙酸胺可捕集過量的 DTT，而防止銀染時(shí)的紋理現(xiàn)象。 100ul樣品緩沖液中10ul20％的碘乙酸胺，并在室溫保溫30min。3）非還原SDS處理：生理體液、血清、尿素等樣品，一般只用 1％SDS沸水中煮3min，未加還原劑，因而蛋白折疊未被破壞，不可作為測定分子量來使用。SDS電泳凝膠中各主要成分的作用？聚丙烯酰胺的作用：丙烯酰胺與為蛋白質(zhì)電泳提供載體， 其凝固的好壞直接關(guān)系到電泳成功與否，與促凝劑及環(huán)境密切相關(guān)；制膠緩沖液：濃縮膠選擇 pH6.7，分離膠選擇pH8.9，選擇tris－HCL系統(tǒng)，TEMED與AP：AP提供自由基，TEMED是催化劑，催化自由基引起的聚合反應(yīng)進(jìn)行；十二烷基硫酸鈉（ SDS）：陽離子去污劑，作用有四：去蛋白質(zhì)電荷、解離蛋白質(zhì)之間的氫鍵、取消蛋白分子內(nèi)的疏水作用、去多肽折疊。提高SDS電泳分辨率的途徑？聚丙烯酰胺的充分聚合，可提高凝膠的分辨率。建議做法：待凝膠在室溫凝固后，可在室溫下放置一段時(shí)間使用。 忌即配即用或4度冰箱放置，前者易導(dǎo)精彩文檔實(shí)用標(biāo)準(zhǔn)文案致凝固不充分，后者可導(dǎo)致 SDS結(jié)晶。一般凝膠可在室溫下保存 4天，SDS可水解聚丙烯酰胺。一般常用的有氨基黑、考馬斯亮藍(lán)、銀染色三種染料，不同染料又各自不同的染色方法，具體可參照郭堯君編著的《蛋白質(zhì)電泳技術(shù)手冊》 P82-103?！拔⑿Α保▋蛇吢N起中間凹下）形帶原因？主要是由于凝膠的中間部分凝固不均勻所致，多出現(xiàn)于較厚的凝膠中。處理辦法：待其充分凝固再作后續(xù)實(shí)驗(yàn)?！鞍櫭肌保▋蛇呄蛳轮虚g鼓起）形帶原因？主要出現(xiàn)在蛋白質(zhì)垂直電泳槽中，一般是兩板之間的底部間隙氣泡未排除干凈。處理辦法：可在兩板間加入適量緩沖液，以排除氣泡。為什么帶出現(xiàn)拖尾現(xiàn)象？主要是樣品融解效果不佳或分離膠濃度過大引起的。處理辦法：加樣前離心；選擇適當(dāng)?shù)臉悠肪彌_液，加適量樣品促溶劑；電泳緩沖液時(shí)間過長，重新配制；降低凝膠濃度。為什么帶出現(xiàn)紋理現(xiàn)象？主要是樣品不溶性顆粒引起的。處理辦法：加樣前離心；加適量樣品促溶劑。什么是“鬼